1. Field of the Invention
The present invention relates to a method for preparing an oligosaccharide fraction. More particularly, it relates to a method for extracting a suitable molecular weight range of oligosaccharides for use, for example, as a substrate for amylase assay.
2. Description of the Prior Art
Heretofore, many processes for preparing maltooligosaccharides have been used. Generally, these involve chromatographic separation by such techniques as partition column chromatography on cellulose, adsorption chromatography on charcoal and exclusion chromatography on polyacrylamide gels or dextran gels. (Whistler et al, JACS 77, 5761 (1955); Whelan et al, Biochem. J. 58, 569 (1954); Whistler et al, JACS 77, 1017 (1955); French et al, JACS 71, 356 (1949); Trenel et al, J. Chrom. 42, 476 (1969); Whistler et al, JACS 72, 677 (1950); Hough et al, J. Chem. Soc. 2511 (1949)) In all of these methods the presence of large amounts of glucose, maltose (G.sub.2) and maltotriose (G.sub.3) severly limits the amount of material that can be fractionated.
Other methods utilize a combination of a fermentation step followed by a chromatographic separation. For example, in U.S. Pat. No. 3,788,910, a brewer's wort is fermented with a yeast to remove sugars of lower molecular weight than those desired. After the yeast is removed, the desired maltotriose/maltotetraose fraction is collected by gel filtration chromatography.
Similarly, studies have shown that commercial baker's yeast is capable of completely removing glucose, maltose and maltotriose but not oligosaccharides of higher weight. Fractionation by column chromatography is required to remove the lower unwanted oligosaccharides such as G.sub.4 and G.sub.5. However, this general method is not very amenable to commercial preparation of large quantities of oligosaccharide fractions of higher molecular weight, such as from about maltopentaose (G.sub.5) to about maltodecaose (G.sub.10). Moreover, since fractionation by column chromatography is required, it is expensive and time consuming.
Because of this inability to prepare oligosaccharides in a commercially feasible and technically acceptable fashion, significant improvements in several analytical systems, which would be possible if such substrates were available, have been prevented. For example, a conventional system for amylase assay incorporates the following reactions:
______________________________________ 1. Starch glycogen or substrate maltodextrin ##STR1## 2. Maltose ##STR2## 3. Glucose + ATP ##STR3## 4. Glucose-6-phosphate + NAD ##STR4## 6-phosphogluconolactone + NADH ______________________________________
The rate of formation of NADH, once zero-order kinetics is established for equation (4), is directly proportional to the amount of amylase present in the sample. NADH is monitored spectrophotometrically by its absorbance at 340 nm.
However, the use of polymeric oligosaccharides such as starch has the distinct disadvantage that a considerable lag is observed before the indicator reaction (Reaction 4) shows zero-order kinetics. Another disadvantage is the relatively low sensitivity of the resultant amylase determination since the hydrolysis of large polymeric oligosaccharides produces many other smaller oligosaccharides rather than predominantly maltose, maltotriose or maltotetraose, which are readily hydrolyzed to glucose by the maltase in Reaction 2, as required. Thus, amylase can cleave several glycosidic bonds in these large polymeric oligosaccharides but no indication of amylase activity will be observed until a proper substrate for maltase activity is produced (Reaction 2). The use of substrates of smaller oligosaccharides which are not hydrolyzed by maltase, such as maltopentaose, maltohexaose, maltoheptaose, maltooctaose, maltononaose or mixtures of these substrates would increase the sensitivity of the test as well as reduce the time required to reach zero-order kinetics of the indicator reaction (Reaction 4), thereby significantly improving the conventional assay. Ideally, pure G.sub.5 would be the preferred substrate since only one G.sub.2 could be produced per glucosidic bond severance, but its cost of production would be commercially prohibitive. The use of the mixture described above, however, would give analytical values for amylase comparable to those obtained if pure G.sub.5 alone were used.